Anti-Cancer Agent

ABSTRACT

The present invention is intended to provide a novel anticancer agent which is effective to a cancer. After administering an agent prepared using burdock fruit extract to a pancreas cancer patient so that a dose of arctigenin was 100 mg or more per day, the tumor reducing effect was observed, and, in addition, lowering of tumor markers was confirmed. The present invention provides an anticancer agent containing arctigenin, wherein a dose of the arctigenin is 100 mg or more per day. In addition, the present invention provides the anticancer agent containing arctigenin and arctiin at a weight ratio of arctigenin/arctiin=0.7 to 1.3.

CROSS REFERENCE

This application is a continuation application of PCT Application No.PCT/JP20131058692 filed Mar. 26, 2013, which claims priority to JapaneseApplication No. 2012-069964 filed Mar. 26, 2002, the contents of whichare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to an anticancer agent containingarctigenin. More particularly, the present invention relates to ananticancer agent containing arctigenin with the dosage per day thereofbeing 100 mg or more.

BACKGROUND ART

A pancreas cancer is one of the refractory cancers, and the 5-yearsurvival rate of the whole pancreas cancer patient is estimated to be 2to 3%. The number of the patients who died from the pancreas cancer hasrapidly increased by approximately 2.5 times for the last 20 years, andthe statistics showed that 26,791 people died from the pancreas cancerin 2009. The incidence and mortality of the pancreas cancer is almostsame number, and the pancreas cancer occupies 6% of causes of the deathfrom cancers in Japan and is fifth place next to lung, stomach, largeintestine and liver in the mortality by the cancer of different parts.

A treatment for the pancreas cancer which is expected to be radical cureis only surgical resection, although approximately 10 to 20% of thewhole pancreas cancer patients can actually undergo the radicalresection, because many patients have been found with the condition of aprogressive cancer (stage III+IV). A median survival period for eachstage is approximately 12 to 30 months for Stage I and II, 9 to 11months for Stage III and around 5 to 6 months for Stage 1V, thereforethe prognosis is very poor and it has been considered that there is alittle possibility of the treatment for the patient, in particular whois unable to undergo the resection.

A gemcitabine has been used as a standard treatment for the progressivepancreas cancer, however other standard treatments have not beenestablished, when the patient became refractory to the gemcitabine. Thecases of good physical status are recognized in the patient with theprogressive pancreas cancer when the patient became refractory to thegemcitabine, and it has been considered that a development of theeffective treatment for such a patient group (pancreas cancer refractoryto gemcitabine) is the important problem in the whole development of thetherapeutics of pancreas cancer.

In recent years, it is reported that cells derived from pancreas cancersuch as PANC-1, AsPC-1, BxPC-1 and KP-3 have strong tolerance to anextreme nutrient starvation state and that elimination of the tolerancemay be a new biochemical approach in cancer therapy (Patent document 1).

It is reported that arctigenin was found to be effective as a materialwhich can cancel the viability of tumor cells under undernutritioncondition in the screening using pancreas cancer cell line PANC-1(Non-patent document 1).

Here, in the Japanese Pharmacopoeia 15th edition, a burdock fruit isdefined as a fruit of burdock, Arctium lappa Linne (Compositae). Inaddition, a burdock fruit is a herbal medicine prescribed forGingyo-san, Kufugedoku-to, Shofusan and the like, and is classified intoa primary material to be used as a pharmaceutical agent exclusively.

A burdock fruit contains approximately 7% of arctiin which is classifiedinto lignan glycoside and approximately 0.6% of arctigenin which is anaglycone of arctiin. According to the above knowledge, it is expectedthat a burdock fruit extract containing arctigenin can be used as ananticancer agent for treating pancreas cancer.

PRIOR ART DOCUMENTS Patent Document

-   Patent document 1: Japanese Kokai Publication No. 2002-065298

Non-Patent Document

-   Non-patent document 1: S. AwaLe, J, Lu, S. K. KaLauni, Y.    Kurashima, Y. Tezuka, S. Kadota, H. Esumi, Cancer Res., 2006, 66    (3), 1751-1757),

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

The present invention is intended to provide a novel anticancer agentwhich has effect on a cancer.

Means to Solve the Problem

A burdock fruit which is known currently contains arctigenin at a lowcontent of approximately 0.6%. In addition, it is hard to dissolve inwater. Therefore, it has been extremely difficult to produce a burdockfruit extract containing arctigenin at high content by a conventionalhot water extraction method.

In addition, it is desired to provide a burdock fruit extract containinga definite content of arctigenin as an active ingredient for use intreatment of pancreatic cancer and the like, however, it is difficult tocontrol the conversion of arctiin into arctigenin so that a definitecontent of arctigenin which is hard to dissolve in water is contained inthe production of the burdock fruit extract containing arctigenin athigh content, as described above.

Furthermore, it is found that a burdock fruit extract containingarctigenin as a major active ingredient as well as containing a definitecontent of arctigenin and arctiin has a particularly excellentanticancer effect when it is used to treat pancreatic cancer and thelike. Therefore, a method for production that can control the content ofarctigenin and arctiin to a definite content is desired in theproduction of a burdock fruit extract containing arctigenin at highcontent. Particularly, a method to make it possible to produce a burdockfruit extract containing arctigenin and arctiin at a weight ratio ofapproximately 1:1 is desired.

As a result of the diligent investigation in order to solve the aboveproblem, the inventors found a technique to make the content ofarctigenin a definite content and a technique to adjust the contentratio of arctigenin to arctiin by adjusting the enzyme activity ofbeta-glucosidase endogenous in a burdock fruit as a raw material, theparticle size of the cut burdock fruit, the temperature whenenzymatically converting arctiin into arctigenin and the temperature inextracting arctigenin and arctiin from the burdock fruit.

In addition, the inventors observed the tumor reducing effect andconfirmed the lowering of the tumor markers, when the agent is preparedat a dosage of 100 mg or more of arctigenin per day using the burdockfruit extract provided by those techniques and administered it to thepatients with pancreas cancer. The present invention was made based onthese surprising findings.

The present invention provides an anticancer agent containingarctigenin, wherein a dose of the arctigenin is 100 mg or more per day.

In addition, the present invention provides the anticancer agent furthercontaining arctiin, wherein a dose of the arctiin is 100 mg or more perday.

In addition, the present invention provides the anticancer agentcontaining arctigenin and arctiin at weight ratio ofarctigenin/arctiin=0.7 to 1.3.

In addition, the present invention provides the anticancer agent,wherein the arctigenin and the arctiin are derived from burdock fruit.

In addition, the present invention provides the anticancer agent fortreating pancreas cancer that is refractory to treatment withgemcitabine.

In addition, the present invention provides an anti cancer stem cellagent containing arctigenin.

In addition, the present invention provides the anti cancer stem cellagent, wherein a dose of the arctigenin is 100 mg or more per day.

Effect of the Invention

The present invention can provide a novel anticancer agent having aneffect on cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D show the results of the thoracoabdominal contrast CT beforeadministering (A); one month later after the beginning of theadministering (B); two month later (C); and three month later (D) thegranules of an example of the present invention.

FIGS. 2A-2D show the results of thoracoabdominal contrast CT beforeadministering (A); one month later after the beginning of theadministering (B); two month later (C); and three month later (D) thegranules of an example of the present invention.

FIGS. 3A-3D show the results of thoracoabdominal contrast CT beforeadministering (A); one month later after the beginning of theadministering (B); two month later (C); and three month later (D) thegranules of an example of the present invention.

FIG. 4 shows the results of the antitumor evaluation in the model animalof the tumor (CAPAN-1 Xenografts).

FIG. 5 shows the results of the antitumor evaluation in the model animalof the tumor (PSN-1 Xenografts).

FIG. 6 shows the results of the PI staining and the pancreas cancer stemcell marker (CD44, CD24 and ESA) staining for cancer stem cell.

FIG. 7 shows the results of the PI staining and the pancreas cancer stemcell marker (CD44 and c-Met) staining for cancer stem cell.

FIG. 8 shows the results of the PI staining and the pancreas cancer stemcell marker staining for cancer stem cell (comparison with the cisplatintreating).

EMBODIMENT FOR CARRYING OUT THE INVENTION

The present invention is described below in detail. The disclosedconditions are examples, and the present invention is not limited tothem.

An anticancer agent of the present invention contains arctigenin as anactive ingredient so that a dose thereof is 100 mg or more per day.Arctigenin may be derived from a plant containing arctigenin, forexample a burdock fruit. Therefore, the anticancer agent of the presentration may contain arctigenin as an active ingredient which is containedin an extract from a plant, for example a burdock fruit extract obtainedfrom a burdock fruit.

Furthermore, the anticancer agent of the present invention may alsocontain arctiin as an active ingredient so that a dose thereof is 100 mgor more per day. Arctiin may be derived from a plant containing arctiin,for example a burdock fruit. Therefore, the anticancer agent of thepresent invention may contain arctiin as an active ingredient which iscontained in an extract from a plant, for example, a burdock fruitextract obtained from a burdock fruit.

The anticancer agent of the present invention may also containarctigenin and arctiin at weight ratio of arctigenin/arctiin=0.7 to 1.3(mole ratio of 1.0 to 1.9). Arctigenin and arctiin may be derived from aplant containing arctigenin and arctiin, for example a burdock fruit.Therefore, the anticancer agent of the present invention may containarctigenin and arctiin which are contained in an extract from a plant,for example a burdock fruit extract, at weight ratio ofarctigenin/arctiin=0.7 to 1.3. Moreover, the anticancer agent maycontain arctigenin so that a dose thereof is 100 mg or more per day andmay contain arctiin so that a dose thereof is 100 mg or more per day.

The plant containing arctigenin and arctiin includes, but is not limitedto, for example Arctium lappa L. (burdock) (sprout, leaf, rhizoma),Carthamus tinctorius (safflower), Centaurea cyanus (cornflower), Cirsiumvulgare, Cnicus benedictus (Centaurea benedicta), Cynara cardunculus(cardoon), Onopordum acanthium, thistle (Aniurokoazami),Forsythia×intermedia, Forsythia ovata, Forsythia suspensa (weepingforsythia), Forsythia viridissima, Sesamum indicum (sesame), Ipomoeacairica, Polygala chinensis, Trachelospermum asiaticum var. glabrum,Trachelospermum asiaticum, Trachelospermum gracilipes var. liukiuense,Trachelospermum gracilipes, Trachelospermum jasminoides var.jasminoides, Trachelospermum jasminoides var. pubescens, Wikstroemiaindica, Persicaria orientalis (Polygonum orientate), Cerasus jamasakura(wild cherry tree), Arabidopsis thaliana, amaranth, Juglans (walnut),Avena sativa (oat), Triticum spelta, soft wheat, Cupressus lusitanica,Torreya nucifera (Japanese nutmeg) and the like. Among them, burdock andweeping forsythia which have high arctigenin content are preferable.

In the present invention, a burdock fruit extract provided by using themethod for producing a burdock fruit extract described below can be usedwhen arctigenin and arctiin are derived from a burdock fruit. Therefore,the productivity at the time of the manufacture can be improved and theanticancer agent can be prepared easily at a low cost. In addition, whena plant except a burdock fruit is used, the extract containingarctigenin and arctiin can be also obtained easily by using method forproducing described below.

The extract powder obtained by using the method for producing a burdockfruit extract described below contains arctigenin and arctiin at weightratio of arctigenin/arctiin=0.7 to 1.3. Therefore, the extract powderobtained by using the method for producing a burdock fruit extract canbe used as an anticancer agent of the present invention having asuperior anticancer effect in comparison with the conventional burdockfruit extract.

Furthermore, the anticancer agent of the present invention can includeany components. For example, the anticancer agent of the presentinvention can be provided in a form including a pharmaceuticallyacceptable base, a carrier, an excipient, a disintegrator, a lubricant,a coloring agent and the like.

Examples of the carrier and the excipient used for the anticancer agentinclude lactose, glucose, saccharose, mannitol, dextrin, potato starch,corn starch, calcium carbonate, calcium phosphate, calcium sulfate,crystalline cellulose and the like.

In addition, examples of the binder include starch, gelatin, syrup, gumtragacanth, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone,hydroxypropyl cellulose, methyl cellulose, ethyl cellulose,carboxymethyl cellulose and the like.

In addition, examples of the disintegrator include starch, agar, gelatinpowder, crystalline cellulose, calcium carbonate, sodium hydrogencarbonate, sodium alginate, sodium carboxymethylcellulose,carboxymethylcellulose calcium and the like.

In addition, examples of the lubricant include magnesium stearate,hydrogenated vegetable oil, talc, macrogol and the like. In addition,any coloring agent permitted to be added to a pharmaceutical agent canbe used as the coloring agent.

In addition, if necessary, the anticancer agent may be coated with oneor more layers using saccharose, gelatin, purified shellac, gelatin,glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, polyvinylpyrrolidone, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, methyl methacrylate,methacrylic acid polymer and the like. In addition, if necessary, a pHregulator, a buffer, a stabilizer, a solubilizer and the like may beadded.

In addition, the anticancer agent can be provided as a formulation inany form. For example, as an orally administered formulation, theanticancer agent can be a tablet such as a sugar-coated tablet, a buccaltablet, a coated tablet or a chewable tablet, a capsule such as atroche, a a powder or a soft capsule, a granule, a suspension, anemulsion, a syrup such as a dry syrup or a liquid formulation such as anelixir.

Alternatively, the anticancer agent can be a formulation foradministration such as intravenous injection, subcutaneous injection,intraperitoneal injection, intramuscular injection, percutaneousadministration, nasal administration, pulmonary administration, enteraladministration, intraoral administration and transmucosal administrationfor parenteral administration. For example, it can be an injectablesolution, a percutaneous absorption tape, an aerosol or a suppository.In addition, the extract powder can be a masked formulation or a filmcoated agent coated with a coating agent, because the extract powder hasa special acrid taste.

Meanwhile, the extract powder provided by using the method for producinga burdock fruit extract described below can be used as it is.

The anticancer agent of the present invention may be prepared by mixingthe purified arctigenin and other components or may be prepared by usingthe burdock fruit extract prepared by the method describe below.

The burdock fruit extract is prepared through a step of cutting a herbalmedicine, an extraction step (an enzymatic conversion step and anextraction step by an organic solvent), a separating step of solid andliquid, a concentrating step and a drying step.

(Process of Cutting Herbal Medicine)

In the method for producing the burdock fruit extract used for theanticancer agent of the present invention, the burdock fruit which is araw material is cut into a size suitable for the extraction. The herbalmedicine which is a raw material is various parts of plant, mineral oranimal or the like with various kinds of size, shape and hardness, andhave to be cut according to the characteristic. The burdock fruit can becut using any means well known to those skilled in the art. For example,a commercial cutting machine can be used.

In the method for producing the burdock fruit extract used for theanticancer agent of the present invention, the activity ofbeta-glucosidase which is an enzyme inherent in the burdock fruit can bemeasured beforehand for the selection of the burdock fruit suitable forthe production of the present invention.

For a method for measuring the activity of beta-glucosidase, forexample, a ground product of a burdock fruit is acted onp-nitrophenyl-beta-D-glucopyranoside (C₁₂H₁₅NO₈: molecular weight301.25) (manufactured by SIGMA-ALDRICH) as a substrate to generatep-nitrophenol, and the enzyme activity can be measured by measuring thevariation of the absorbance at 400 nm. For the unit representing theenzyme activity, the enzyme level generating 1 μmol of p-nitrophenol perminute can be expressed as 1 unit (U).

In order to provide the burdock fruit extract containing arctigenin andarctiin at a weight ratio of arctigenin/arctiin=0.7 to 1.3, a burdockfruit can be used wherein the activity of the beta-glucosidase which isinherent in the burdock fruit is 0.4 U/g or more, preferably 1 U/g ormore. If the activity is lower than 0.4 U/g, the hydrolysis will beinsufficient and the weight ratio of arctigenin will decrease, andtherefore the desired burdock fruit extract cannot be obtainedefficiently.

In addition, in the method for producing the burdock fruit extract usedfor the anticancer agent of the present invention, a burdock fruit whichis cut into an arbitrary particle size can be used. The smaller theparticle size of the cut burdock fruit is, the more the enzymaticconversion may be promoted and the yield of the extract may beincreased. On the other hand, if the particle size is too small, thecontrol of the process may become difficult because the enzymaticconversion is too fast, and a trouble may occur for the accuratesolid-liquid separation in the subsequent step.

In order to provide the burdock fruit extract containing arctigenin andarctiin at a weight ratio of arctigenin/arctiin=0.7 to 1.3, as shown inthe following EXAMPLES, the burdock fruit is cut into a particle size of9.5 mm or less, for example, the burdock fruit is cut so that all of theburdock fruit particles pass through a sieve of 9.5 mm.

In addition, in order to provide the burdock fruit extract containingarctigenin and arctiin at a weight ratio of arctigenin/arctiin 0.7 to1.3, it is desirable that the burdock fruit is cut so that all of theburdock fruit particles pass through a sieve of 9.5 mm and, for example,60 to 100% are distributed on a sieve of 0.85 mm, and more preferably,65 to 80% are distributed on a sieve of 0.85 mm.

(Extraction Step)

The extraction step is the most important step in terms of quality amongsteps of producing a powder of the herbal medicine extract. Due to thisextraction step, the quality of the powder of the herbal medicineextract is decided. In the method for producing the burdock fruitextract of the present invention, two stages of the enzymatic conversionstep and the extraction step using an organic solvent are carried out toextract the burdock fruit extract.

(Enzymatic Conversion Step)

The enzymatic conversion step is an important step in the method forproducing the burdock fruit extract used for the anticancer agent of thepresent invention. The enzymatic conversion step is a step ofenzymatically converting arctiin contained in the burdock fruit intoarctigenin by beta-glucosidase which is an enzyme in the burdock fruit.

Specifically, the cut burdock fruit prepared by the above steps isretained at an appropriate temperature to make the beta-glucosidasefunction and progress the reaction from arctiin to arctigenin. Forexample, an arbitrary solution such as water is added to the cut burdockfruit and stirred at a temperature such as around 30° C., and therebythe burdock fruit can be retained at an arbitrary temperature.

In order to provide the burdock fruit extract containing arctigenin andarctiin at a weight ratio of arctigenin/arctiin=0.7 to 1.3, the cutburdock fruit is retained at a temperature of around 30° C., such as atemperature between 20 to 50° C.

If it is lower than 20° C., the hydrolysis will be insufficient and theweight ratio of arctigenin will decrease, and therefore the desiredburdock fruit extract cannot be obtained efficiently. On the other hand,if it is er than 50° C., the enzyme will be deactivated and the weightratio of arctigenin will be decrease, and therefore the desired burdockfruit extract cannot be obtained efficiently.

In addition, the length of time for retaining is not limitedparticularly, as far as the burdock fruit is retained at the abovetemperature, and for example, it can be retained for approximately 30minutes. By retaining at 20 to 50° C., an appropriate amount of arctiinis enzymatically converted into arctigenin regardless of the length oftime for retaining, and the burdock fruit extract containing arctigeninand arctiin at a weight ratio of approximately 1:1 can be obtained.

(Extraction Step Using Organic Solvent)

The extraction step using an organic solvent is a step of extractingarctigenin and arctiin from the burdock fruit using any appropriateorganic solvent. In other words, it is a step of extracting the burdockfruit extract by adding an appropriate solvent in the state in which thecontent of arctigenin is increased after the above step of enzymaticconversion. For example, an appropriate solvent is added to the burdockfruit extract, and the mixture is heated and stirred for an appropriatelength of time to extract the burdock fruit extract. In addition, theburdock fruit extract can be extracted using any extraction method wellknown to those skilled in the art such as a method of heating to reflux,a method of drip extraction, a method of dipping extraction or a methodof pressurized extraction, other than the heating and stirring method.

Because arctigenin is hardly soluble in water, the yield of arctigenincan be improved by adding an organic solvent. Any organic solvent can beused as the organic solvent. For example, alcohols such as methanol,ethanol and propanol as well as acetone can be used. In consideration ofsafety, ethanol is preferably used as the organic solvent in the methodfor producing the burdock fruit extract used for the anticancer agent ofthe present invention.

If the burdock fruit extract is extracted by the heating and stirring,the heating and stirring can be carried out at any temperature, however,the burdock fruit is retained at a temperature of 80° C. or more, suchas a temperature between 80 to 90° C. in order to provide the burdockfruit extract containing arctigenin and arctiin at a weight ratio ofarctigenin/arctiin=0.7 to 1.3.

In addition, the length of time for the heating and stirring is notlimited particularly, as far as the burdock fruit is heated and stirredat the above temperature, and arctigenin and arctiin can be extractedfrom the burdock fruit into the solvent by the heating and stirring theburdock fruit for approximately 30 minutes, such as 30 to 60 minutes.

The longer the length of time for the heating and stirring is, the morethe yield of arctigenin and arctiin is improved. However, if the lengthof time for the heating and stirring is longer, larger amounts ofunnecessary oils and fats begin to dissolve, and the load of theconcentrating step becomes larger. Therefore, the length of time for theheating and stirring should be determined appropriately according to thesituation.

In addition, the larger the amount of ethanol is, the higher thesolubility of arctigenin and arctiin and the more the yield ofarctigenin and arctiin is improved. However, if the amount of ethanol islarger, larger amounts of unnecessary oils and fats begin to dissolve,and the load of the concentrating step becomes larger. Therefore, theadded amount should be determined appropriately according to thesituation. Note that the burdock fruit extract can be sterilized andpasteurized simultaneously by the heating and stirring in this step.

(Separating Step of Solid and Liquid)

The step of separating solid and liquid is a step of separating theburdock fruit subjected to the extraction from the extract liquid. Thesolid-liquid separation can be carried out using any method well knownto those skilled in the art. The method for solid-liquid separationincludes, for example, a filtration method, a sedimentation method and acentrifugal separation method. Industrially, a centrifugal separationmethod is desirable.

(Concentrating Step)

The concentrating step is a step of removing the solvent from theburdock fruit extract liquid prior to drying. The removal of the solventfrom the burdock fruit extract liquid can be carried out using anymethod well known to those skilled in the art.

However, it is preferable for the extract liquid from the burdock fruitprovided by the above steps not to be exposed furthermore to a hightemperature for a long time.

For example, the burdock fruit extract liquid can be concentrated byusing a decompression concentration method without being exposed to ahigh temperature for a long time.

The burdock fruit extract liquid can be concentrated until the burdockfruit extract with a desired concentration can be obtained.

For example, it is desirable to concentrate the extract liquid to theextent that the drying can be carried out appropriately in the followingdrying step. Alternatively, when the burdock fruit extract is dried andformulated into a powder in the following steps, it is desirable toconcentrate the extract liquid until appropriate properties of theformulation can be obtained.

Because arctigenin and arctiin are hardly soluble in water, a largequantity of arctigenin and arctiin may adhere to the inside of themanufacturing apparatus in the following drying step, and thereby thefinal yield may be largely decreased. Therefore, dextrin can be added tothe burdock fruit extract liquid provided in the concentrating step inorder to prevent the adhesion of arctigenin and arctiin to themanufacturing apparatus. For example, the amount of the dextrin to beadded is preferably around 15 to 30% relative to the solid content ofthe concentrated liquid.

(Drying Step)

It is a step of finishing the burdock fruit extract provided by theabove steps into a powder form. The drying can be carried out using anymethod well known to those skilled in the art. For example,freeze-drying and spray drying are known as a drying method, and it iscommon to use the former for laboratory scale production and the latterfor mass production.

The burdock fruit extract containing arctigenin and arctiin at a weightratio of arctigenin/arctiin=0.7 to 1.3 can be provided by the aboveproduction process. The method for producing the burdock fruit extractmust include a step of enzymatic conversion at a temperature from 20° C.to 50° C., however it is not necessary to include all of the othersteps.

In addition, the burdock fruit extract containing arctigenin at highconcentration can be provided easily at low cost by using theabove-mentioned production process. Therefore, the anticancer agent ofthe present invention can be prepared easily at low cost by using theburdock fruit extract provided by using this method.

In addition, the arctigenin concentration of the burdock fruit extractprovided by the above-mentioned production process is high, so that thetotal weight of the anticancer agent per day can be reduced incomparison with the case when the conventional burdock fruit extract isused. Therefore, the burden of a patient can be reduced.

Note that, the present invention also provides a method for producing aburdock fruit extract containing arctigenin and arctiin at a weightratio of arctigenin/arctiin=0.7 to 1.3 (mole ratio of 1.0 to 1.9),including steps of: cutting a burdock fruit, and converting arctiinwhich is inherent in the burdock fruit into arctigenin by enzymaticconversion by beta-glucosidase which is inherent in the burdock fruit,wherein the enzymatic conversion includes a reaction at a temperaturefrom 20° C. to 50° C.

In addition, the present invention provides the method for producing theburdock fruit extract, wherein the burdock fruit is cut into a particlesize from 0.85 mm to 9.5 mm in the step of cutting.

Furthermore, the present invention provides the method for producing theburdock fruit extract, wherein the enzyme activity of thebeta-glucosidase which is inherent in the burdock fruit is 0.4 U or moreper 1 g of the burdock fruit.

In addition, the present invention provides the method for producing theburdock fruit extract, including a step of extracting an extractcontaining arctigenin and arctiin by adding an organic solvent after thestep of enzymatic conversion.

In addition, the present invention provides the method for producing theburdock fruit extract, wherein the organic solvent is ethanol.

In addition, the present invention provides the method for producing theburdock fruit extract, wherein the extract is extracted at approximately80° C. in the extraction step.

Furthermore, the present invention provides a burdock fruit extractcontaining arctigenin and arctiin at weight ratio ofarctigenin/arctiin=0.7 to 1.3 which is provided by the above method.

Furthermore, the present invention provides an anticancer agentcontaining the burdock fruit extract containing arctigenin and arctiinat weight ratio of arctigenin/arctiin=0.7 to 1.3 which is provided bythe above method.

The present invention can provide an anticancer agent with antitumoreffect which contains arctigenin so that a dose thereof is 100 mg ormore per day. Moreover, the present invention can provide an anticanceragent containing arctigenin and arctiin at weight ratio ofarctigenin/arctiin=0.7 to 1.3. Stable inhibition of the growth of atumor and the antitumor effect can be expected by administering theanticancer agent of the present invention to a patient suffering fromcancer, for example pancreas cancer. In addition, the anticancer agentof the present invention can be used as a therapeutic agent for pancreascancer to treat pancreas cancer that is refractory to treatment withgemcitabine.

In addition, test example 7 below showed that the toxicity of thegranule containing arctigenin and arctiin was very low. Therefore, theanticancer agent of the present invention can provide high anticancereffect with very few side effects.

In addition, test examples 8 and 9 below showed that arctigenin has aneffect of killing pancreas and hepatic cancer stem cell. Therefore, itis suggested that arctigenin demonstrates anticancer activity to solidcancer by acting on and killing not only tumor body but also cancer stemcell. Moreover, it showed that arctigenin has an anticancer effect bykilling not only pancreas cancer stem cell but also other cancerouscancer stem cell. Therefore, the present invention provides ananticancer agent for the treatment of various cancers.

The present invention also provides an anti cancer stem cell agentcontaining arctigenin. A cancer stem cell refers to a cell having aproperty of a stem cell among cancer cells.

The anti cancer stem cell agent of the present invention can be composedsame as the anticancer agent of the present invention described above.Therefore, the anti cancer stem cell agent may contain arctigenin sothat a dose thereof is 100 mg or more per day. In addition, the anticancer stem cell agent may further contain arctiin or may containarctiin so that a dose thereof is 100 mg or more per day. The anticancer stem cell agent may contain arctigenin and arctiin at a weightratio of arctigenin/arctiin=0.7 to 1.3. Arctigenin and arctiin may bederived from a plant containing arctigenin and arctiin, for example,burdock fruit.

The test examples 8 and 9 below showed that arctigenin has an effect ofkilling cancer stem cell selectively, particularly under the nutritionstarvation condition which is close to the environment around cancercell. Therefore, the anti cancer stem cell agent of the presentinvention can inhibit the growth of cancer cell by killing cancer stemcells and therefore can be preferably used as an anticancer agent.

EXAMPLES Test Examples

The influence on arctigenin/arctiin ratio (weight ratio) caused by theenzyme activity of the burdock fruit and the condition of the enzymaticconversion (temperature and length of time), namely, the cause andeffect relationship of both, was verified.

(Measurement of Enzyme Activity)

Burdock fruits differing in production regions and lots were pulverizedby a Wiley mill, and 0.1 g of each pulverized burdock fruit was dilutedwith 10 mL of water as a sample solution.

As a substrate solution, water was added to 0.15 g ofp-nitrophenyl-beta-D-glucopyranoside and the volume was fixed at 25 mLto prepare a 20 mmol/L aqueous solution ofp-nitrophenyl-beta-D-glucopyranoside. 0.5 mL of the 20 mmol/L aqueoussolution of p-nitrophenyl-beta-D-glucopyranoside was added to 1 mL of a0.1 mol/L acetate buffer to prepare a cocktail and the cocktail waspreheated at 37° C. for approximately 5 minutes.

0.5 mL of the sample solution was added to the cocktail and they werereacted at 37° C. for 15 minutes, and then 2 mL of a 0.2 mol/L aqueoussolution of sodium carbonate as a reaction stop solution was added tostop the reaction. The absorbance of this solution at 400 nm wasmeasured and the enzyme activity was calculated based on the variationfrom the blank solution that was not subjected to the enzymatic reactionusing the following formula.

Enzyme activity (U/g)=(Absorbance of the sample solution−Absorbance ofthe blank solution)×4 mL×1/18.1 (Millimolar molecular extinctioncoefficient of p-nitrophenol under the above measurement condition:cm²/μmol)×1/Light path length (cm)×1/Reaction time (minute)×1/0.5mL×1/Sample solution level (g/mL)

It was confirmed that the enzyme activity of each burdock fruit was 0.12to 8.23 U/g as shown in Table 1.

Test Example 1

7 mL of water was added to 1 g of the cut burdock fruit having an enzymeactivity of 0.12, 0.27 or 0.40 U/g (Samples 1 to 3), and the temperatureconditions for the enzymatic reaction were set to 15° C. and 20° C. andthe reaction time at respective reaction temperature was set to 30minutes. Ethanol was added after the reaction, and the extraction wascarried out at 80° C., arctigenin and arctiin of the obtained extractwere quantitated, and the weight ratio of arctigenin/arctiin wascalculated. The results are shown in Comparative Examples 1 to 2 andExample 1 of Table 1.

In Sample 3 having an enzyme activity of 0.40 U/g, a burdock fruitextract with arctigenin/arctiin (weight ratio)=0.82 was obtained whenthe temperature of the enzymatic reaction was 20° C. and the reactiontime was 30 minutes.

On the other hand, arctigenin/arctiin (weight ratio) was 0.69 when thetemperature of the enzymatic reaction was 15° C. and the reaction timewas 30 minutes, therefore the temperature of the enzymatic reaction ispreferably 20° C. or more.

In addition, Samples 1 and 2 having an enzyme activity of less than 0.40U/g could not satisfy arctigenin/arctiin (weight ratio)=0.70 or moreeven if the temperature of the enzymatic reaction was 20° C., thereforethe enzyme activity of the burdock fruit is preferably 0.40 U/g or more.

Test Example 2

7 mL of water was added to 1 g of the cut burdock fruit having an enzymeactivity of 4.03 U/g (Sample 5), the temperature conditions for theenzymatic reaction were set to 30° C., 40° C., 50° C. and 60° C. and thereaction time at respective reaction temperature was set to 15 minutesand 30 minutes (only 30° C. and 60° C.). The extraction was carried outusing ethanol after the reaction, and arctigenin and arctiin of theobtained extract were quantitated, and the weight ratio ofarctigenin/arctiin was calculated.

The results are shown in Example 3 of Table 1. The burdock fruitextracts with arctigenin/arctiin (weight ratio)=0.7 when the temperatureof the enzymatic reaction was 30° C. and the reaction time was 15minutes, with arctigenin/arctiin (weight ratio)=1.0 when the temperatureof the enzymatic reaction was 30° C. and the reaction time was 30minutes, with arctigenin/arctiin (weight ratio)=1.2 when the temperatureof the enzymatic reaction was 40° C. and the reaction time was 15minutes, and with arctigenin/arctiin (weight ratio)=1.2 when thetemperature of the enzymatic reaction was 50° C. and the reaction timewas 15 minutes, were obtained.

On the other hand, arctigenin/arctiin (weight ratio) was 0.4 when thetemperature of the enzymatic reaction was 60° C. and the reaction timewas 15 minutes and arctigenin/arctiin (weight ratio) was 0.5 when thetemperature of the enzymatic reaction was 60° C. and the reaction timewas 30 minutes. Based upon the foregoing, the temperature of theenzymatic reaction is preferably less than 60° C.

Test Example 3

7 mL of water was added to 1 g of the cut burdock fruit having an enzymeactivity of 1.42 U/g (Sample 4), the temperature condition for theenzymatic reaction was set to 25° C. and the reaction time was set to 10minutes and 30 minutes. The extraction was carried out using ethanolafter the reaction, and arctigenin and arctiin of the obtained extractwere quantitated, and the weight ratio of arctigenin/arctiin wascalculated.

The results are shown Example 2 of Table 1. The burdock fruit extractswith arctigenin/arctiin (weight ratio)=0.74 when the temperature of theenzymatic reaction was 25° C. and the reaction time was 10 minutes andwith arctigenin/arctiin (weight ratio)=0.85 when the temperature of theenzymatic reaction was the same and the reaction time was 30 minutes,were obtained.

Based upon the foregoing, even if the enzyme activity was 1.42 U/g,desired results were able to be obtained.

TABLE 1 Comparative Comparative example 1 example 2 Example 1 Example 2Example 3 Example 4 Example 5 sample Sample 1 Sample 2 Sample 3 Sample 4Sample 5 Sample 6 Sample 7 enzyme activity (U/g) 0.12 0.27 0.40 1.424.03 7.82 8.23 arctigenin/ 15° C. 30 min 0.26 0.50 0.69 — — — — arctiin20° C. 30 min 0.23 0.60 0.82 — — — — (weight 25° C. 10 min — — — 0.74 —— — ratio) 30 min — — — 0.85 — — — 30° C. 15 min — — — — 0.70 — — 30 min— — — — 1.00 0.93 0.89 40° C. 15 min — — — — 1.20 — — 30 min — — — — — —— 50° C. 15 min — — — — 1.20 — — 30 min — — — — — — — 60° C. 15 min — —— — 0.40 — — 30 min — — — — 0.50 — —

Example 6 Production of Burdock Fruit Extract 1

A burdock fruit (enzyme activity was 8.23 U/g) was cut, and all theparticles passed through a sieve of 9.5 mm were further passed through asieve of 0.85 mm and confirmed that 75% of them were remained. 80 kg ofthis chopped burdock fruit was added to 560 L of water kept warm at 29to 33° C. and stirred for 30 minutes. Then, 265 L of ethanol was addedand the temperature was raised to 85° C. and the mixture was furtherheated to reflux for 60 minutes. This solution was centrifuged and aburdock fruit extract liquid was obtained. This operation was repeatedtwo times and the obtained extract liquids were combined, and vacuumconcentration was carried out, and 20% of dextrin relative to the solidcontent of the extract was added and the mixture was spray dried. Thearctigenin content and the arctiin content were 6.2% and 7.1%respectively, and the burdock fruit extract powder witharctigenin/arctiin (weight ratio)=0.89 (containing 20% of dextrin) wasobtained.

Example 7 Production of Burdock Fruit Extract 2

A burdock fruit (enzyme activity was 8.23 U/g) was cut, and all theparticles passed through a sieve of 9.5 mm were further passed through asieve of 0.85 mm and confirmed that 75% of them were remained. After 80kg of this chopped burdock fruit was added to 560 L of water kept warmat 30 to 33° C. and stirred for 30 minutes, 265 L of ethanol was addedand the temperature was raised to 85° C., and the mixture was furtherheated to reflux for 30 minutes. This solution was centrifuged and aburdock fruit extract liquid was obtained. This operation was repeatedtwo times and the obtained extract liquids were combined, and vacuumconcentration was carried out, and 20% of dextrin relative to the solidcontent of the extract was added and the mixture was spray dried. Thearctigenin content and the arctiin content were 6.0% and 6.8%respectively, and the burdock fruit extract powder witharctigenin/arctiin (weight ratio)=0.87 (containing 20% of dextrin) asobtained.

Example 8 Production of Burdock Fruit Extract 3

A burdock fruit (enzyme activity was 7.82 U/g) was cut, and all theparticles passed through a sieve of 9.5 mm were further passed through asieve of 0.85 mm and confirmed that 75% of them were remained. 80 kg ofthis chopped burdock fruit was added to 560 L of water kept warm at 30to 32° C. and stirred for 40 minutes, and after 60 minutes, 258 L ofethanol was added and the temperature was raised to 85° C., and themixture was further heated to reflux for 30 minutes. This solution wascentrifuged and a burdock fruit extract liquid was obtained. Thisoperation was repeated two times and the obtained extract liquids werecombined, and vacuum concentration was carried out, 20% of dextrinrelative to the solid content of the extract was added and the mixturewas spray dried. The arctigenin content and the arctiin content were6.2% and 6.7% respectively, and the burdock fruit extract powder witharctigenin/arctiin (weight ratio)=0.93 (containing 20% of dextrin) wasobtained.

Example 9 Production of Burdock Fruit Extract 4

A burdock fruit (enzyme activity was 7.82 U/g) was cut, and all theparticles passed through a sieve of 9.5 mm were further passed through asieve of 0.85 mm and confirmed that 75% of them were remained. After 80kg of this chopped burdock fruit was added to 560 L of water kept warmat 30 to 32° C. and stirred for 30 minutes, 253 L of ethanol was addedand the temperature was raised to 85° C. and the mixture was furtherheated to reflux for 40 minutes. This solution was centrifuged and aburdock fruit extract liquid was obtained. This operation was repeatedtwo times and the obtained extract liquids were combined, and vacuumconcentration was carried out, 25% of dextrin relative to the solidcontent of the extract was added and the mixture was spray dried. Thearctigenin content and the arctiin content were 6.4% and 7.2%respectively, and the burdock fruit extract powder witharctigenin/arctiin (weight ratio)=0.89 (containing 25% of dextrin) wasobtained.

TABLE 2 Example 6 Example 7 Example 8 Example 9 burdock enzyme 8.23 8.237.82 7.82 fruit activity step of particle size 0.85-9.5 0.85-9.50.85-9.5 0.85-9.5 cutting (mm) step of temperature 29 to 33° 30 to 33°30 to 32° 30 to 32° enzymatic (° C.) C. C. C. C. conversion time (min)30 min 30 min 40 min 30 min extraction solvent ethanol ethanol ethanolethanol step temperature 85° C. 85° C. 85° C. 85° C. (° C.) time (min)60 min 30 min 30 min 40 min step of separating Centrif- Centrif-Centrif- Centrif- solid and liquid ugal ugal ugal ugal separationseparation separation separation concentrating step vacuum vacuum vacuumvacuum concen- concen- concen- concen- tration tration tration trationdrying step spray spray spray spray drying drying drying dryingarctigenin/arctiin 0.89 0.87 0.93 0.89 (weight ratio)

From the results of the above Examples 6 to 9, it was found that aburdock fruit extract with the content of arctigenin:arctiin (weightratio)=approximately 1:1 can be provided by enzymatically convertingarctiin into arctigenin at approximately 30° C. in the step of enzymaticconversion. A reaction caused by an enzyme usually progresses dependenton the temperature and the length of time, however, it was found that,at the above temperature, a burdock fruit extract with the content ofarctigenin:arctiin (weight ratio)=approximately 1:1 can be obtainedregardless of the length of time for the enzymatic conversion.

In addition, from the results of the above Examples 6 to 9, it was foundthat a burdock fruit extract with the content of arctigenin:arctiin(weight ratio)=approximately 1:1 can be obtained by raising thetemperature to approximately 85° C. for heating to reflux in the step ofheating to reflux. Usually, when the heating to reflux is carried out toobtain an extract, the amount of the ingredient within the extractchanges dependent on the temperature and the length of time, however, itwas found that, at the above temperature, a burdock fruit extract withthe content of arctigenin:arctiin (weight ratio)=approximately 1:1 canbe obtained regardless of length of time for heating to reflux.

Example 10 Granule Formulated with Burdock Fruit Extract Powder

(1) Burdock fruit extract powder of Example 7 33.3% (2) Lactose 65.2%(3) Hydroxypropyl cellulose  1.5% Total  100%

(Method for Production)

A granule was produced according to the paragraph of Granule in theGeneral Rules for Preparations of “Japanese Pharmacopoeia”. That is,ingredients from (1) to (3) described in the above list were taken andproduced into a granule form. Each 1.5 g of the granular material waspacked into an aluminum laminate film, and a granule containing 0.5 g ofthe burdock fruit extract powder per one package was obtained.

Example 11 Granule Formulated with Burdock Fruit Extract Powder

(1) Burdock fruit extract powder of Example 7 66.7% (2) Lactose 30.3%(3) Hydroxypropyl cellulose  3.0% Total  100%

(Method for Production)

A granule was produced according to the paragraph of Granule in theGeneral Rules for Preparations of “Japanese Pharmacopoeia”. That is, theingredients from (1) to (3) described in the above list were taken andproduced into a granule form. Each 3.0 g of the granular material waspacked into an aluminum laminate film, and a granule containing 2 g ofthe burdock fruit extract powder per one package was obtained.

Example 12 Tablet Formulated with Burdock Fruit Extract Powder

(1) Burdock fruit extract powder of Example 7 37.0% (2) Crystallinecellulose 45.1% (3) Carmellose calcium 10.0% (4) Crospovidone  3.5% (5)Hydrous silicon dioxide  3.4% (6) Magnesium stearate  1.0% Total  100%

(Method for Production)

A tablet was produced according to the paragraph of Tablet in theGeneral Rules for Preparations of “Japanese Pharmacopoeia”. That is, theingredients from (1) to (6) described in the above list were taken andproduced into the tablet.

Test Example 4

The effect on pancreas cancer was examined using the granule of Example10.

The patient (53 years old, man), who had been confirmed that he has anadenocarcinoma (including adenosquamous carcinoma) by histopathology andthen Gemcitabine+S-1 therapy had been carried out as a pre-treatmentduring one year and three months, and then Best-response had been aPartial Response (PR) and then the cancer had finally acquiredresistance to Gemcitabine+S-1 therapy and then became PD (ProgressiveDisease), was targeted. The 7.5 g (5 packs) of the granule of Example 10(containing 2.5 g of burdock fruit extract powder) was dailyadministered orally to this patient after breakfast once a day.

Note that, 1 g of the burdock fruit extract powder which was containedin the granule used in this test contained 59.4 mg of arctigenin and68.5 mg of arctiin. In other words, 148.5 mg of arctigenin and 171.25 mgof arctiin were administered to the patient per day.

FIGS. 1A-1D to FIGS. 3A-3D show the images of a thoracoabdominalcontrast CT in this patient before administering the granules (A); onemonth later after the beginning of the administering (B); two monthlater (C); and three month later (D). The evaluation of the tumorreducing effect was carried out time dependently from these images basedon new guidelines for therapy effect evaluation on solid cancer (RECISTguideline (version1.1)). As a result, the tumor reducing effect wasconfirmed as shown in arrows in FIGS. 2A-2D and 3A-3D. On the otherhand, no serious side effect after administration of the granule ofExample 10 was seen in the above patient.

Furthermore, quantity of tumor markers CA19-9 and CEA before and afteradministration of the granule was examined. As a result, the drop of thetumor markers CA19-9 and CEA after administration of the granule wasconfirmed as shown in Table 3.

TABLE 3 one month two month three month before later after later afterlater after admin- admin- admin- admin- istering istering isteringistering CA19-9 (U/mL) 69.7 172.5 123 100.6 CEA (ng/mL) 66.4 224.1 114.898.3

Therefore, it has been shown that the anticancer agent of the presentinvention is effective to pancreas cancer. Especially, it is shown thatit is effective to pancreas cancer that is refractory to treatment withgemcitabine. It has not been known conventionally that the compositionof the present invention is effective to pancreas cancer whenadministrated, and additionally, the effect could not be achieved easilyunless the composition was actually administered to a person.

In addition, it can be expected that the anticancer agent of the presentinvention is effective to not only pancreas cancer but also hypovascularcancer in hypoxia and uncernutrition state and the like, such as coloncancer.

Test Example 5

Three pancreas cancer patients who had been refractory to gemcitabinetherapy carried out as pre-treatment were targeted and were dailyadministered orally with 3 g of the granule of Example 10 (containing 1g of the burdock fruit extract powder) after breakfast once a day. Inother words, 59.4 mg of arctigenin and 68.5 mg of arctiin wereadministered to the patients per day.

As a result, the tumors of these patients did not increase for 1 to 2months after the beginning of the administering, however they werefinally decided as PD. Therefore, neither tumor reducing effect norlowering of the tumor markers was confirmed. In addition, no seriousside effect after administration of the above granule was seen in any ofthe patients.

Example 12 Production of Burdock Fruit Extract by Cutting, ColdExtraction and Ethanol Addition

The 200 g of chopped burdock fruit was added to 1 L of water (22° C.)and stirred for an hour, added by 0.45 L of ethanol and further heatedto reflux for an hour. It was filtered by 4 sheets of gauze (100 meshwire netting) and washed with 0.5 L of 30% ethanol, and the combinedextraction solution (1.5 L) was freeze-dried. The burdock fruit extractwhich was obtained by cold extraction using cutting of herbal medicinecontained 13.3% of arctigenin and 11.4% of arctiin respectively witharctigenin/arctiin (weight ratio)=0.86.

Test Example 6 Anticancerous Evaluation in Tumor Model Animal

(Method for Test)

The model animal of a tumor was made by seeding human pancreas cancercell line CAPAN-1 or PSN-1 in subcutis of back of a nude mouse(BALB-cAJnu/nu; CLEA Japan, Inc.) as a donor and transplanting the tumormass of the provided donor mouse into subcutis of back of a recipientmouse, Arctigenin (AG), arctiin (A) and the burdock fruit extract(Example 12) were dissolved in DMSO in concentration of 10 mg/ml,diluted with a saline, and then administered orally in stomach at 50 μgper one mouse five times a week. The anticancerous was evaluated bymeasuring the size of the subcutaneous tumor mass of the back with time.

The remarkable inhibiting effect on tumor growth was seen in the agentadministered group compared with control in one month after thebeginning of the administering. The antitumor effect was also obtainedin the group administered with purified arctigenin, however, thestronger antitumor effect was seen in the burdock fruit extract (Example12) containing the precursor arctiin together (FIGS. 4 and 5). It wasconfirmed that the burdock fruit extract with arctigenin/arctiin (weightratio)=0.7 to 1.3 showed higher antitumor activity.

Test Example 7 Expression Frequency of Dose Limiting Toxicity (DLT)

By targeting the 15 pancreas cancer patients who had been refractory togemcitabine therapy carried out as pre-treatment, the phase I study ofthe granule containing arctigenin was carried out to investigate theexpression frequency of dose limiting toxicity. These 15 patients weredaily administered orally with predetermined dose of the granulecontaining arctigenin after breakfast once a day. Three of these 15patients were administered with 3 g of the granule of Example 10(containing 1 g of the burdock fruit extract powder, namely 59.4 mg ofarctigenin and 68.5 mg of arctiin) per once. Another three of these 15patients were administered with 7.5 g of the granule of Example 10(containing 2.5 g of the burdock fruit extract powder namely 148.5 mg ofarctigenin and 171.25 mg of arctiin) per once. Still another ninepatients were administered with 12 g of the granule of Example 10(containing 4 g of the burdock fruit extract powder namely 237.6 mg ofarctigenin and 274 mg of arctiin) per once.

As a result, among these 15 targeted patients, the expression frequencyof dose limiting toxicity was 0. Specifically, the main adverse eventsof Grade 3 or more in the phase I study of the granule containingarctigenin against the pancreas cancer patient were a rise of GGT,hyperglycemia, a rise of the ALP and a rise of blood bilirubin, andtherefore no serious adverse event was observed.

TABLE 4 Adverse event in the phase I study of the granule containingarctigenin (n = 15, during all course periods) Item G1 G2 G3 G4 % G 3-4Subjective and objective symptom Diarrhea 3 1 0 0 0 Nausea 2 2 0 — 0Stomachache 1 0 0 — 0 Vomiting 3 0 0 0 0 Inappetence 3 4 0 0 0Laboratory values Leukopenia 5 0 0 0 0 Neutropenia 1 0 0 0 0 Anemia 11 20 0 0 Thrombocytopia 3 0 0 0 0 Blood bilirubin 1 1 1 0 6.67 increase ASTincrease 9 2 0 0 0 ALT increase 6 1 0 0 0 ALP increase 10 1 1 0 6.67 GGTincrease 4 0 7 0 46.7 Cr increase 0 1 0 0 0 Hyperglycemia 5 6 4 0 26.7

It was found from these result that the granule of the Examples had verylow toxicity and high safety. Therefore, it has been shown that thepresent invention can provide an anticancer agent with fewer sideeffects.

Test Example 8 Effect on Pancreas Cancer Stem Cell

Then, the effect of arctigenin on cancer stem-like cells (CSCs), whichwas considered to be deeply involved in various treatment resistance,recurrences and appearance of metastases in cancer, was examined.

(Preparation of Medium and Reagent)

For preparation of the glucose-containing medium, 4.75 g of Dulbecco'smodified Eagle medium 2 (Nissui Pharmaceutical) was dissolved in water,added by 12.5 ml of 1M HEPES pH 7.4 (DOJINDO LABORATORIES, 342-01375),sterilized, added by 18.5 ml of 10% NAHCO₃, 10 ml of L-glutamine(Sigma-Aldrich), 5 ml of Anti-Anti (Life technologies), 5 ml of MEMNON-ESSENTIAL AMINO ACID SOLUTION (Sigma-Aldrich) and 50 ml of FETALBOVINE SERUM (Biowest) that was inactivated in a warm bath at 56° C. for30 minutes, and finally filled up to 500 ml.

The glucose inhibition medium was prepared by adding 2-Deoxy-Glucose(2-DG) (Tokyo Chemical Industry) in 20 mM of final concentration to theglucose-containing medium.

The medium containing 3 μM arctigenin was prepared by adding arctigenin(Kracie Pharmaceutical) in 3 μM of final concentration to theglucose-containing medium or the glucose inhibition medium.

For preparation of FACS buffer, 10 g of Bovine serum albumin Proteasefree (Wako Pure Chemical industries) was dissolved in 1 L of PBS (−),added by sodium azide in 0.1% of final concentration and performedfiltration sterilization.

For the fluorescence-labeled antibody for FACS assays, CD44 (338803 or338807, BioLegend), CD24 (311117, BioLegend), ESA (324205, BioLegend)and c-Met (11-8858, eBioscience) were used. In addition, a staining stepwas carried out according to the attached data sheet to the product.

(Method for Test and Results)

The pancreas cancer cell PANC-1 (ATCC No CRL-1469 was seeded in aglucose-containing medium, incubated overnight and then incubated ineach of the glucose-containing medium, the glucose inhibition medium,the glucose-containing medium containing 3 μM arctigenin and the glucoseinhibition medium containing 3 μM arctigenin for 24 hours. Aftercollected cells, PI staining (dead cell staining) and cancer stem cellmarker staining were carried out according to the usual method and anAnalysis was carried out using flow cytometry (FACS). For a marker,triple positive of CD44, CD24 and ESA (CD326) or double positive of CD44positive and c-Met strong positive which were reported as a stem cellmarker of pancreas cancer were used.

FIG. 6 shows the results of P1 staining and cancer stem cell marker(triple positive of CD44⁺, CD24⁺ and ESA⁺ (CD326)) staining. As a resultof PI staining, the cellular survival rate was 78.20% for theglucose-containing condition, 68.53% for the glucose inhibitioncondition and 69.50% for the glucose-containing condition in thepresence of 3 μM arctigenin, whereas the survival rate was 35.71% forthe glucose inhibition condition in the presence of 3 μM arctigenin.

In addition, as a result of the cancer stem cell marker staining, ratio(the number of the survival) of CD44⁺ESA⁺CD24⁺ cell which indicated apancreas cancer stem cell was 4.41% (441) for the glucose-containingcondition, 6.51% (651) for the glucose inhibition condition and 5.01%(501) for the glucose-containing condition in the presence of 3 μMarctigenin, whereas 0.98% (98) for the glucose inhibition condition inthe presence of 3 μM arctigenin in all analyzed cells. Therefore, it wasshown that arctigenin has an effect of killing pancreas cancer steincell under the glucose starvation condition.

FIG. 7 shows the results of PI staining and cancer stem cell marker(double positive of CD44⁺ and c-Met^(High)) staining. As a result of PIstaining, the cellular survival rate was 84.70% under theglucose-containing condition, 88.80% under the glucose inhibitioncondition and 83.30% under the presence of 3 μM arctigenin in theglucose-containing condition, whereas the survival rate was 27.50% underthe presence of 3 μM arctigenin in the glucose inhibition condition.

The positive ratio (the number of the survival) of CD44 positive c-Metstrong positive (CD44⁺,c-Met^(High)) cell, which was reported one afteranother as a new marker of pancreas cancer stem cell using a strongtumorigenicity as an indicator was 0.63% (38) for the glucose-containingcondition, 0.78% (47) for the glucose inhibition condition and 0.52%(31) for the glucose-containing condition in the presence of 3 μMarctigenin, whereas 0.22% (13) for the glucose inhibition condition inthe presence of 3 μM arctigenin in all analyzed cells. Therefore, it hasbeen shown that arctigenin has an effect of killing pancreas cancer stemcell under a glucose starvation condition. Therefore, it has been shownthat arctigenin also has an effect of killing pancreas cancer stem cellthat expresses these stern cell markers under the glucose starvationcondition.

Test Example 9 Comparative Test with Preexisting Chemotherapeutic AgentAgainst Pancreas Cancer Stem Cell

The effect of killing pancreas cancer stem cell of arctigenin wascompared with that of cisplatin which was one of the preexistingchemotherapeutic agents against pancreas cancer.

(Preparation of Medium and Reagent)

For preparation of the glucose-containing medium, 4.75 g of Dulbecco'smodified Eagle medium 2 (Nissui Pharmaceutical) was dissolved in water,added by 12.5 ml of 1M HEPES pH 7.4 (DOJINDO LABORATORIES, 342-01375),sterilized, added by 18.5 ml of 10% NAHCO₃, 10 ml of L-glutamine(Sigma-Aldrich), 5 ml of Anti-Anti (Life technologies), 5 ml of MEMNON-ESSENTIAL AMINO ACID SOLUTION (Sigma-Aldrich) and 50 ml of FETALBOVINE SERUM (Biowest) that was inactivated in a warm bath at 56° C. for30 minutes, and finally filled up to 500 ml.

The glucose inhibition medium was prepared by adding 2-Deoxy-Glucose(2-DG) (Tokyo Chemical industry) in 15 mM of final concentration to theglucose-containing medium.

The medium containing 4 μM arctigenin was prepared by adding arctigenin(Kracie Pharmaceutical) in 4 μM of final concentration to theglucose-containing medium or the glucose inhibition medium.

(Method for Test and Results)

The pancreas cancer cell Capan-1 (ATCC No. HTB-79) was seeded in aglucose-containing medium, incubated overnight and then incubated ineach of a glucose-containing medium, a glucose inhibition medium, aglucose-containing medium containing 4 μM arctigenin and a glucoseinhibition medium containing 4 μM arctigenin, or in 7 μM cisplatin(CDDP: Wako Pure Chemical Industries) for 24 hours. After collectedcells, PI staining (dead cell staining) and cancer stem cell markerstaining were carried out according to the usual method and an Analysiswas carried out using flow cytometry (FACS). For the marker, triplepositive of CD44, CD24 and ESA (CD326) which was reported as a sterncell marker of pancreas cancer was used.

FIG. 8 shows the results of P1 staining and cancer stem cell markerstaining. As a result of PI staining, the cellular survival rate was67.57% for the glucose-containing condition, 79.10% for the glucoseinhibition condition and 72.80% for the glucose-containing condition inthe presence of 4 μM arctigenin, whereas the survival rate was 48.73%for the glucose inhibition condition in the presence of 4 μM arctigeninand 58.08% under the cisplatin treatment.

In addition, as a result of the cancer stem cell marker staining, theratio (the number of the survival) of CD44⁺ESA⁺CD24⁺ cell which isindicative of the pancreas cancer stem cell was 0.87% (65) for theglucose-containing condition, 1.40% (105) for the glucose inhibitioncondition and 1.04% (78) for the glucose-containing condition in thepresence of 4 μM arctigenin, whereas 0.24% (18) for the glucoseinhibition condition in the presence of 4 μM arctigenin and 1.17% (88)for the cisplatin treatment in all analyzed cells. On the other hand,when they were analyzed in viable cells, 1.28% for theglucose-containing condition, 1.77% for the glucose inhibition conditionand 1.43% for the glucose-containing condition in the presence of 4 μMarctigenin, whereas 0.50% for the glucose inhibition condition in thepresence of 4 μM arctigenin, and it rose to 2.02% for the cisplatintreatment. Therefore, it has been shown that arctigenin has an effectfor killing CD44⁺ESA⁺CD24⁺ positive pancreas cancer stem cell which isdifficult to be killed by cisplatin under the glucose starvationcondition.

It has been strongly suggested from the results of the test examples 8and 9 that arctigenin has the effect of killing cancer stem-like cellsin the nutrition starvation condition which is close to the environmentwhere cancer cell is placed in the body. In addition, it has beenstrongly suggested that arctigenin has not only the effect of inhibitingthe growth of cancer cell by acting on tumor body but also the effect ofacting on and killing cancer stem cell which can supply cancer cell andreconstruct tumor tissue.

In addition, the granule containing arctigenin in the test example 7showed very low toxicity. Arctigenin has the killing effect in thenutrition starvation condition and acts on not only cancer body cell butalso cancer stem cell, so that it is suggested that arctigenin can lowerthe influence on normal cell and may acquire the low toxicity.Therefore, the present invention can provide an anticancer agent havinghigh anticancer activity with few side effects.

INDUSTRIAL APPLICABILITY

The present invention can be preferably used for an anticancer agentespecially a pancreas cancer therapeutic agent.

What is claimed is:
 1. A composition for treating cancer comprising theanticancer agent arctigenin, wherein said arctigenin is present in anamount of 100 mg.
 2. The composition of claim 1, wherein saidcomposition is a pharmaceutical composition.
 3. A method of treatingcancer comprising administering to a patient in need thereof, a dose ofarctigenin of at least 100 mg per day.
 4. The composition of claim 1 orclaim 2, wherein said composition further comprises arctiin.
 5. Themethod of claim 3, further comprising administering to said patient adose of arctiin of at least 100 mg per day.
 6. The composition of claim4, wherein said composition comprises arctigenin and arctiin at a weightratio of arctigenin/arctiin=0.7 to 1.3.
 7. The composition of claim 1 orclaim 2, wherein said arctigenin and said arctiin are derived fromburdock fruit.
 8. The method of claim 3 or claim 5, wherein said canceris refractory to treatment with gemcitabine.
 9. The composition of claim1 or claim 2, wherein said cancer is refractory to treatment withgemcitabine.
 10. A composition for inhibiting cancer stern cellcomprising arctigenin.
 11. The composition of claim 10, wherein saidcomposition is a pharmaceutical composition.
 12. The composition ofclaim 10 or claim 11, wherein said arctigenin is present in an amount ofat least 100 mg.
 13. The composition of claim 10 or claim 11, whereinsaid arctigenin is present in an amount of 100 mg.
 14. A method ofinhibiting cancer stem cells comprising administering to said patient acomposition comprising arctigenin at a dose of at least 100 mg or moreper day.